The general goal of this project is to determine the dynamic organization and regulation of specific cytoskeletal components in living cells, using a combination of chemical probes, imaging techniques, and biophysical analyses. Experiments in the next funding period will start with the development of three new probes that should expand our abilities to study molecular functions in vivo: a probe that reveals cell adhesion forces, a probe that depolymerizes actin filaments upon photoactivation, and a probe that facilitates the detection of single molecules. These probes will then be applied in conjunction with existing methods including fluorescent analog cytochemistry, computer image restoration, photobleaching, and resonance energy transfer imaging to study molecular interactions during cell locomotion, cell-substrate adhesion, and cytokinesis. Experiments will be performed to determine the distribution and magnitude of traction forces under moving fibroblasts, the corresponding organization of contractile structures, and the regulatory roles of protein phosphorylation and microtubules. In addition, the assembly of actin will be studied at focal adhesions, where it might play a role in maintaining structural integrity and in force generation. The dynamics of vinculin will be examined in relation to a putative regulatory mechanism involving conformational changes. Experiments will also be performed to probe the mechanism of cytokinesis, by mapping the contribution of different regions of the cortex in generating cleavage forces, and by determining the spatial relationship between microtubles and the cortex. These experiments should provide crucial basic knowledge for understanding not only normal physiological functions, such as embryonic development and wound healing, but also a number of diseases related to cell locomotion or division including birth defects and cancer.